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Cite this: Phys. Chem. Chem. Phys.,
2015, 17, 15534
Protein synthesis in artificial cells: using
compartmentalisation for spatial organisation
in vesicle bioreactors†
Received 17th December 2014,
Accepted 17th March 2015
Yuval Elani,ab Robert V. Lawab and Oscar Ces*ab
DOI: 10.1039/c4cp05933f
www.rsc.org/pccp
Whereas spatial organisation of function is ubiquitous in biology, it
has been lacking in artificial cells. We rectify this by using multicompartment vesicles as chassis for artificial cells, allowing distinct
biological processes to be isolated in space. This is demonstrated by
in vitro synthesis of two proteins in predefined vesicle regions.
Artificial cells are constructs that have been built from the
bottom up using biological or synthetic building blocks, which
mimic biological cells in morphology or function. In bottom-up
synthetic biology lipid vesicles are widely used as cellular models
and as chassis for artificial cells. They have been used to host a range
of biological and biomimetic processes, including reproduction,1
biomolecule synthesis,2 cell–cell communication,3,4 and sense/
response behaviours.5 They have been utilized as mimics of
multicellular assemblies6–8 and as simplified models of biomembranes, allowing insights into the potential role of
membrane biophysics in modulating cellular processes.9–11
Vesicles are considered an attractive platform for such applications due to their biological compatibility, their ability to host
transmembrane proteins, their rich phase behaviour, and their
resemblance of cellular membranes.
One of the defining characteristics of biological cells is their
spatial organisation of content and function, with different biochemical processes confined to specific cellular regions. In contrast,
artificial cells have had largely homogenous interiors. In order for
artificial cells to start mimicking the complexity of their biological
counterparts, and for them to exhibit higher order properties, this
divide needs to be bridged. Herein we use compartmentalisation as
an engineering principle to rectify this: multi-compartment vesicles
are used as chassis for artificial cells that have specific regions
devoted to different biological processes.
a
Department of Chemistry, Imperial College London, Exhibition Road, SW7 2AZ,
UK. E-mail: [email protected]
b
Institute of Chemical Biology, Imperial College London, Exhibition Road,
SW7 2AZ, UK
† Electronic supplementary information (ESI) available: Experimental methods.
See DOI: 10.1039/c4cp05933f
15534 | Phys. Chem. Chem. Phys., 2015, 17, 15534--15537
We have previously developed methods to construct vesicles
with defined compartment number, content and size, where
compartments were separated from one another via a lipid
bilayer.12 We also used such multi-compartment vesicles as microreactors that hosted a multi-step reaction cascade, mimicking a
cellular signalling pathway.13 This work builds on this: we demonstrate protein synthesis using in vitro transcription and translation
(IVTT), using the separation of content to achieve segregation
of function. We show for the first time that each compartment
can be devoted to the biochemical synthesis of distinct biomolecules—green fluorescent protein (GFP) being synthesised
in one compartment, red fluorescent protein (RFP) in another
(Fig. 1). In doing so we succeed in using compartmentalisation
to introduce a level of spatial organisation of function that has
not been previously seen in artificial cells.
For cell-free protein expression we used the PURExpress14
IVTT kit that contained the biochemical components necessary
to synthesise proteins in the presence of a plasmid. The reaction
mix was made by adding the kit components to plasmids encoding
for fluorescent proteins, as well as an added volume to yield a final
solution composition of 0.9 M sucrose and 9 mM 1,2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) liposomes (see ESI† for
experimental details). Two such solutions were made: one
containing Dasher-GFP plasmid, the other an RFP plasmid.
Two-compartment vesicles were generated according to previously published methods, using the phase transfer of two
water-in-oil droplets (Fig. 1 and ESI† for details).12,13 Briefly, a
water/oil column was first made, with a 4.7 mM DPhPC lipid-inoil solution. This was left to stabilise for two hours, resulting
in a densely packed monolayer at the water/oil interface. Two
sucrose-loaded lipid-stabilised droplets were then expelled
above the same location of the column: one droplet containing
the GFP plasmid, the other the RFP plasmid. As these droplets
descended through the column under gravity, they made contact
with another resulting in a droplet interface bilayer. As the droplet
pair entered the aqueous phase of the column, they were enveloped
by a second monolayer, encasing both droplets together in a
bilayer, and resulting in dual-compartment vesicles with defined
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Fig. 1 (A) A schematic of the vesicle-based artificial cell, with each compartment dedicated to the synthesis of a particular fluorescent protein. (B) Twocompartment vesicles were constructed using the phase transfer of two lipid-coated water-in-oil droplets through an interfacial monolayer of an oil/
water column. Dotted arrows represent the movement of droplets under gravity as they are expelled from the tubing, sink through the column, and
transformed into vesicles. Images not to scale.
content in each compartment. Immediately after vesicles were
generated, 1.5 M sucrose was injected into the bottom of the
aqueous phase to reduce the osmotic imbalance between the
vesicle interior and exterior.
Protein synthesis was followed over time by monitoring
fluorescence using a FITC filter for GFP and a TRITC filter for
RFP at 100 ms and 1000 ms exposures respectively (Fig. 2). Over
the course of 50 minutes at 37 1C, fluorescence of both
compartments was observed to increase concurrently, indicating successful spatially segregated protein synthesis: different
regions of the vesicle were specialised to perform distinct
biological processes, acting as modules within the artificial cell
system. Furthermore, as biological macromolecules are largely
impermeable to membranes, the process remained spatially
isolated over the course of the experiment.
Vesicles were found to be formed with a lower efficiency
than those previously reported, with approximately 12% successful rate of formation (n = 50). This was attributed to the
presence of a relatively large concentration of macromolecules
(estimated at 2.6 mg ml 1 by the supplier), which are thought
to interfere with the formation of a well-packed monolayer at
the water/oil interface of the droplet precursors. Friddin et al.
observed the same effect in a droplet interface bilayer system
and specifically attributed it to the energy supply fraction of
the IVTT kit,15 which destabilised the interface. In a similar
manner to our experiments, they (and others)16 counteracting
this by adding liposomes inside the droplets to increase the
probability of sufficient monolayer coverage.
On the occasions where phase transfer was unsuccessful,
vesicles were observed to degrade in one of two ways (discussed
in detail elsewhere).12 Either the inner bilayer failed, leading
to fusion of the two compartments, or the external bilayer
failed whereupon the compartment content was released to
the external solution.
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Fig. 2 (A) Schematic and brightfield images showing the transformation
from a droplet pair in an oil environment to a two-compartment vesicle
surrounded by a bulk aqueous solution. Note the contrast difference in the
three different stages. (B) Brightfield–fluorescent composite images of
a spatially segregated artificial cell at different times after they were
generated. Cell-free protein synthesis from template DNA was successful,
with GFP (green channel) synthesised in one region, RFP (red channel) in
another. (C) Brightfield images of several two-compartment vesicles that
were generated. As previously noted, oil pockets were seen to accumulate
on the vesicle surface.12 Scale bar = 200 mm.
The above IVTT experiments had to be carried out in different
conditions to those commonly used and recommended, specifically in the presence of sucrose and lipids. To test the effect of
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Finally, in this work we extend the concept of soft-matter
compartmentalisation to the field of artificial cells.31 Compartmentalisation is a recurring feature in chemical, biological, and
engineering systems. It refers to the splitting-up of a unit into
distinct spatial sub-divisions, each one considered a module
designed separately and optimised to perform a designated task,
often in different chemical environments. This enables multi-part,
higher-order, or emergent features to arise, and leads to more
efficient systems. Our approach differs from bulk self-assembly
approaches31 with respect to the greater degree of control over
vesicle properties (compartment content, number, and size) and
that only one bilayer separates compartments from one another.
There is therefore scope for engineering communication routes
between compartments, for example by the insertion of transmembrane proteins or DNA origami pores.32
Fig. 3 Comparison of the effect different additives have on the efficiency
of GFP IVTT, after 2 hours of incubation at 37 1C. Error bars = S.D, n = 5.
these additives, we measured GFP expression levels on a fluorimeter with and without these components present. The kit
was assembled as before, incubated at 371 for two hours, and
fluorescence levels then recorded (Fig. 3). These results
revealed that the presence of lipid did not have a detrimental
effect on IVTT efficiency. In contrast, the presence of sucrose
decreased the reaction efficiency, possibly due to sucrose being
a source of RNase contamination.
In this work, we monitored successful IVTT of different proteins
in different compartments via the production of fluorescent proteins. Others have adopted similar approaches to investigate various
features of cell-free expression in vesicles, including the stochastic
nature of IVTT,17 the effect of vesicle volume on the reconstitution of
transmembrane proteins,18 the presence of feedback loops,19 for
programmable triggering of IVTT via vesicle fusion,20 for the in vitro
evolution of protein pores,21 and for quantitative and statistical
insights into proteins synthesis rates.22,23
This work demonstrates the suitability of the multicompartment vesicle platform to host and effectively compartmentalise distinct, complex biochemical systems such as IVTT,
thus further establishing its use as a chassis for vesicle-based
artificial cells. This is made possible by the 100% encapsulation
efficiency and the ability to finely control the number of
compartments and their contents. The method developed thus
complements alternative compartmentalisation strategies—
including vesicles-in-vesicles,24,25 phase separated vesicles,26,27
and tether-linked vesicles28,29—that have previously proved
powerful in a microreactor context.
The fact that our vesicles are larger than biological cells
render them unsuitable for certain applications in their current
form. However, there are intense ongoing efforts surrounding
microfluidic generation of vesicles and related membrane-based
structures that enable scale-down in size and a scale-up in
generation throughput, which could in principle help facilitate
a shift towards functional use in vivo.30
15536 | Phys. Chem. Chem. Phys., 2015, 17, 15534--15537
Conclusions
We generated two-compartment vesicles, with two ‘genomes’
contained in each compartment. Using compartmentalisation
as an engineering principle allowed protein synthesis of distinct
proteins to occur in defined regions, and demonstrated the spatial
segregation of complex processes in a vesicle-based artificial cell.
This has implications in microreactor and medicinal settings, for
instance for isolated on-board synthesis multiple therapeutic
biomolecules in a cell-like drug-delivery vehicle, and can be
considered an enabling technology for bottom up synthetic
biology. Finally, the developed system extends the concept of
modularisation that is central to synthetic biology.33,34 Instead
of applying to genetic circuits, here it pertains to modular
processes that take place within individual compartments of
an artificial cell. This paves the way higher-order cellular
characteristics to be introduced in future.
This work was supported by the EPSRC via grants EP/J017566/1,
EP/K038648/1, EP/G00465X/1, BBSRC Grant BB/F013167/1, by an
EPSRC Centre for Doctoral Training Studentship from the Institute
of Chemical Biology (Imperial College London) awarded to Y.E,
and by an EPSRC Doctoral Prize Fellowship awarded to Y.E.
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